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Biocontrol Potential of Bacillus strains against soybean cyst nematode (Heterodera glycines) and for promotion of soybean growth

BMC Microbiology volume 24, Article number: 371 (2024) Cite this article

Abstract

The soybean cyst nematode (SCN, Heterodera glycines) is the most yield-limiting pathogen in soybeans worldwide. Using chemical pesticides to control this disease is harmful to human and environment. It is urgent to develop environment-friendly nematicides. The aim of this study was to discover novel biocontrol agents on H. glycines control and soybean growth under greenhouse and field conditions Eight Bacillus strains were isolated from soil rhizosphere soils and the stability and efficiency of H. glycines was assessed in greenhouse and field experiments in 2021 and 2022. In particular, the Ba2-6 strain had the highest potential, because it was a biocontrol agent against H. glycines shown to cause 93.85% juvenile mortality. Furthermore, strains Ba 1–7, Ba2-4, and Ba2-6 effectively reduced the number of females and improved the soybean seed number per plant. Based on their morphological, physiological, biochemical and molecular (16 S rRNA) characteristics, the three strains were identified as B. aryabhattai (Ba1-7), B. megatherium (Ba2-4), and B. halotolerans (Ba2-6). The ability of Ba2-6 to induce systemic resistance to H. glycines in soybeans was investigated by the split-root system and real-time quantitative PCR experiments. The results indicated that the Ba2-6 strain induced systemic resistance to suppress the penetration of H. glycines, and enhanced gene expression of PR1, PR3a, PR5, and NPR1-2, involved in the salicylic acid and jasmonic acid pathways. The study suggests that the strains of B. aryabhattai Ba1-7, B. megatherium Ba2-4, and B. halotolerans Ba2-6 can be considered as effective biocontrol agents to control H. glycines. Further, B. halotolerans Ba2-6 not only promotes soybean growth but also enhances resistance to H. glycines by regulating defense-related gene expression and inducing systemic resistance in soybean.

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Introduction

Soybean (Glycine max), one of the important oil crops in the world, has a crucial role in balancing the output and quality of global agriculture every year. However, Soybean cyst nematode (Heterodera glycines, SCN) is the most harmful pathogen of soybean (Glycine max (L.) Merr.) worldwide. Indeed, the nematode is a sedentary endoparasite, and has been detected in the most soybean growing regions around the world, annually inducing the yield loss of 5-90% [1,2,3]. H. glycines not only directly causes soybean damage but also interacts with other plant pathogens to cause complex infections, making it difficult to control. H. glycines invades plants as second-stage juveniles (J2) and establishes feeding sites to obtain nutrients from soybean plants, which leads to the decrease of soybean nodulation and nitrogen fixation, and causes typical symptoms of weak roots and the early yellowing of plants [4]. In China, it has been estimated that the soybean loss caused by H. glycines can exceed 120 million USD every year [5].

At present, chemical nematicides are still the main method for the management of SCN. However, considering human health and environmental pollution, many highly toxic chemical nematicides have been restricted use in China [6]. Furthermore, newly developed and released chemical nematicides such as fluensulfone and fluopyram, have drawbacks such as longer residual periods in soil and higher prices compared to traditional nematicides) [7,8,9]. Seed treatment of biocontrol strains is a method for controlling nematode diseases. Under the influence of nematodes, biocontrol strains can induce plant resistance to nematodes and promote plant growth. Therefore, it is urgent to develop environment-friendly nematicides and find a simple and effective approach to control plant parasitic nematodes. Thus, the seed coating approach, which is effective in controlling plant diseases and pests minimizes pesticide dust. Seed treatment is used as a standard method for the application of biocontrol bacteria that could simultaneously confer resistance to nematodes and promote plant growth in plant production [10, 11], and induce the expression of some resistance related genes involved in salicylic acid, jasmonic acid, and ethylene pathways, such as GmPR1, GmPR2, GmPR5, GmNPR1, and GmSAMT [12, 13]. Therefore, developing alternative safe and efficient method such as biocontrol is urgent for SCN control.

Microorganisms as biocontrol agents are more eco-friendly than chemical pesticides, as biocontrol agents have been widely used to control harmful pests and pathogens [14]. Indeed, bacteria are examples of typical biocontrol agents, such as Pseudomonas spp. and Bacillus spp., because some bacteria are safe, natural, selective, and friendly for environmental and human health. Recently, several bacterial products (Bacillus firmus I-1582, Burkholderia spp. A396) are commercially available to control plant-parasitic nematodes (PPN) [15, 16]. In addition, the potential of a commercial-grade strain of rhizobacteria as a biocontrol agent has also well been evaluated [17]. Bacteria can reduce nematodes in infested plants through direct and/or indirect antagonistic mechanisms, highlighting that biological control of nematodes is feasible.

Recently, many bacteria including Bacillus spp., Pseudomonas spp., and Sinorhizobium spp. have shown the potential for the biocontrol of PPN [11, 18, 19]. Among them, Bacillus is widely used because of its wide distribution and strong inter-genus or inter-species competitiveness competition. Importantly, Bacillus can not only improve the control against nematodes but also promote plant growth [20,21,22]. Indeed, it is very important for biocontrol strains to produce antagonistic components against nematodes. For example, B. aryabhattai Sneb517 exhibited antagonistic activity to H. glycines juveniles, leading to a significant suppression of egg hatch [22]. Similarly, plant treatment with B. subtilis Sneb 815, also exhibited efficacy in reducing the number of galls in both the pot experiment and field condition relative to the control [11]. In addition, the mixed application of different Bacillus could control plant parasitic nematode diseases [21]. Therefore, Bacillus is a good biocontrol agent that has great potential to control plant parasitic nematodes.

Biocontrol of PPN is attributed to bacteria directly or indirectly affecting nematodes through producing secondary metabolites or inducing plant systemic resistance [23]. Studies have shown that there are different mechanisms for the biological control ability of Bacillus, such as the production of iron carriers, ammonia, and IAA, secretion of hydrolases, ISR, antibacterial activity, and the response of plants to accumulate pathogenesis-related (PR) proteins [24,25,26]. Previous studies showed that the cuticle of nematodes can prevent the organisms from being damaged by the environment, which is a very hard and non-perishable multilayer exoskeleton [27, 28]. Also, bacterial proteases can degrade and destroy the cuticle of the nematodes [29,30,31]. Thus, these evidences indicate that the production of proteases is involved in the prevention and control of nematodes. Furthermore, Salicylic acid (SA), and/or jasmonic acid (JA)/ethylene (ET) pathways are a key component in plant defense. The PR1, PR2, and PR5 genes are markers of the SA signaling pathway, PR3b is involved in the JA/ET signaling pathway, and SA-induced defense responses are mediated by NPR1 [32,33,34,35]. For instance, Bacillus megaterium Sneb207 have displayed their potential to induce systemic resistance in soybean and increase the expression of GmNPR1-1 [13]. In addition, split-root analysis is an important method used to confirm ISR to nematodes caused by biocontrol strains [18].

The objective of the present study was to (1) isolate/screen biological agents and evaluate the effect of the selected Bacillus strains on the control of H. glycines under both greenhouse and field conditions for two years, (2) identify the selected isolate strains, and (4) investigate the ability of Ba2-6 to induce systemic resistance to H. glycines in soybean and produce proteases.

Materials and methods

Isolation of Bacillus

This study site was located at Anda Science and Technology Park of Heilongjiang Bayi Agricultural University, Anda, Heilongjiang, China. Different bacterial isolates were obtained from the rhizosphere in an experimental soybean field to screen the resistance potential to soybean cyst nematode (46°24’E, 125°20’N). Bacillus isolates were selected with a heat treatment. Briefly, ten grams of soil were suspended in 90 mL of sterile distilled water, shaken for 10–20 min to prepare soil suspension and then incubated in a water bath at 80 °C for 10 min. 100 µLof soil suspension with concentration gradients of 10−4, 10−5, and 10−6 were evenly applied on a Nutrient Agar (NA) plate. Each concentration of which was repeated three times. The plates were incubated at 28 ± l °C for 24–48 h, then classified and purified according to the size, color, and shape of the colony. Well-isolated colonies were maintained on the NA plate for 72 h. After that, prepare the bacterial suspension were prepared and incubated in a water bath at 80 °C for 10 min [36, 37]. Based on the morphological characteristics of the strains, 76 strains of Bacillus subtilis were saved and preserved. Based on the results of the larvicidal efficacy in vitro, 8 strains were selected for further analysis.

Soybean cyst nematodes

A population of H. glycines (race 3, HG type 0) was used in this study. Nematodes were collected from the experimental field of the Daqing branch of Heilongjiang Academy of Agricultural Sciences, Daqing, Heilongjiang, China. The cysts in the soil were separated by the elutriation screening method. Cysts were then surface-sterilized with 0.5% (w/v) NaClO for 3 min and rinsed several times with sterile distilled water [19]. After surface disinfection, the cysts were incubated in a self-made incubator. The incubator was made of centrifuge tubes and gauze, and used after sterilization. Then put the incubator at 26 ± l°C in the dark, and hatched second-stage juveniles (J2) were collected every 24 h and stored at 4˚C for use [22].

Evaluation of larvicidal efficacy in vitro

The nematicidal effects of Bacillus isolates were evaluated against SCN under laboratory conditions. A pure single colony was inoculated in 100 mL beef extract-peptone liquid medium (NB) in a flask and shaken at 150 rpm for 48 h at 28 ± l °C on a rotary shaker. Using sterile water to adjust the final concentration of fermentation broth to about 108 CFU mL−1. The supernatant was filtered through a 0.22-µm cell-free filter [22]. A 500-µL fermentation filtrate was shifted to a 1 mL sterilized Petri dish containing approximately 100 freshly hatched H. glycines J2. NB liquid medium was used as a control. Separated sets of Petri dishes were maintained for 48 h for observation. Then, the dead J2s (no movement) were confirmed by touching the juvenile with a fine needle and counted under a microscope [21]. Five replicates were conducted for each treatment and the entire experiment was repeated twice.

Evaluation of the nematicidal ability of the selected bacterial isolates in the pot experiment

Soybean [Glycine max (L.) Merr. ‘Hefeng50’] was grown in the experimental field of the Heilongjiang Bayi Agricultural University. Soybean variety-Hefeng50 was provided by the Daqing branch of the Heilongjiang Academy of Agricultural Sciences (Heilongjiang, China), which is susceptible to SCN. The soybean seeds were sterilized with 0.5% sodium hypochlorite, and then rinsed with sterilized water multiple times and air-dried.

The seeds were coated with the selected bacterial fermentation broth (1.0*108 cfu/mL) at a 70:1 mass ratio. Control soybean seeds were coated with sterilized water. All seeds were sown in plastic pots (9 cm × 9 cm) with the sterilized sand soil mixture (1:1, v/v) and grown in a greenhouse at 26/21°C with a 16/8 h photoperiod. Then, when soybeans grew to the two-leaf stage, approximately 2,000 active J2s were dispensed in 8 mL of water and distributed evenly into the soil around the seedling through three 1-cm deep holes [21]. On the 35th day after nematode inoculation, the plants were carefully removed from their respective pots, and the number of cysts on the roots was counted. Then the plants were washed, blotted dry, and weighed. In each case, there were five replicates, and the entire experiment was repeated twice.

Evaluation of the nematicidal ability of the selected bacterial isolates under field conditions

Field experiments were conducted in the soybean fields naturally infested by H. glycines race 3 at Anda city (46°41′ N, 125°35′ E) during the growing seasons of 2021 and 2022. Hefeng50 seeds were coated with either the selected bacterial fermentation broth or NB (control), as described above. However, seeds coated with sterile water were used for a non-treated control. Then the seeds of each treatment were sown in different units. Each experimental unit (2. 8 m×5 m) consisted of a five-row plot planted with 100 soybean seeds per row. The treatments were arranged as a randomized complete block design, and each treatment included five units.

On the 30th day after soybean emergence, 30 seedlings were randomly selected from the inner three rows of each plot, and the number of cysts on soybean roots was investigated. The shoot length, taproot length, and fresh weight of the soybean were determined. In autumn, 30 seedlings were also selected as above from each unit, pod numbers per plant and seed numbers per plant were measured. The seeds were taken back to the laboratory for threshing, and then the weight of 100 different seeds was measured three repeats for each unit.

Identification of bacterial isolates

According to the results of greenhouse and field tests, three strains were selected from the eight strains for identification. Using Bergey’s Manual of Determination Bacteriology [38], the three screened bacterial strains were identified at the genus level by using morphological and chemical taxonomic characteristics. Benson’s method was used for gram staining [39]. Microtubes for chemotaxonomic and biochemical reactions were obtained from Qingdao Haibo Biotechnology Company, China. The ribosomal DNA isolated from the bacteria was amplified via PCR with universal bacterial primers 27 F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492R (5′- ACG GCT ACC TTG TTA CGA CTT-3′). The reaction mixture was 10 µL 2 × Taq PCR master mix (TIANGEN Company, Beijing, China), 1 µL of template DNA (50 ng/µL), 1 µL each of forward and reverse primers (10 µM), and 7 µL of double-distilled water [11]. The PCR was performed following the method reported by Kumar [40]. The PCR products were carried out on a PCR thermal cycler (Bio-Rad, Hercules, California, USA), analyzed by electrophoresis on 1.2% (wt/vol) agarose Gels, and sent to Jilin kumei Biotechnology Co., Ltd. for sequencing.

The obtained sequences were analyzed by BLASTn (NCBI; http://blast.ncbi.nlm.nih.gov). The sequences of related species and genera were obtained from the GenBank database, and the phylogenetic research was carried out by the neighbor-joining method at 1000 replications with MEGA version 7 [41]. The sequences were lined up by Clustal W and analyzed by using the maximum likelihood method reported by Saito [42, 43].

Determination of protease production capacity of Ba2-6 strain

Concurrently, we investigated the production of protease by strain Ba2-6. Protease activity was determined by the modified method, as being described before [44, 45], using casein as substrate. Briefly, we aliquoted 10 µL of bacterial liquid (108 CFU/mL) onto the sterilized single-layer filter paper, and placed the filter paper in the casein medium plate. Here, the Ba2-6 strain was repeated 3 to 4 times. After incubation at 37 ℃ for 24 h, the size of the transparent circle around the colony on the plate was observed, and the plate was submerged with 10% HCl solution for judgment. We finally measured the diameter of the colony and the transparent circle, and then took the mean value and calculated the ratio of transparent circle diameter to the colony diameter. The higher ratios indicated smaller colonies.

Effect of Ba2-6 on inducing systemic resistance to H. glycines in soybean

Soybean seeds were surface-sterilized as described earlier and planted in pots containing the sterilized sand soil mixture, with one seed per pot. When soybeans produced two euphylla, the roots were removed from the pots and washed with flowing sterile water. Then, roots with consistent growth were selected and divided into two equal parts with a scalpel, each of which was replanted in separate pots and labeled as inducer and responder, respectively [46]. When the two parts of the root systems had been successfully fixed, the inducers were inoculated either with 5 mL of the Ba2-6 suspension (108 CFU mL−1) or sterilized water as a control. Five days later, all responder roots were inoculated with 1,000 J2 of H. glycines. Ten days after nematode inoculation, the responder roots were stained with NaClO-acid fuchsin [47], and the number of nematodes inside the soybean roots as counted under a microscope. Each treatment had four repetitions, and the entire experiment was repeated twice.

Real-time qRT-PCR analysis

To demonstrate whether Ba2-6 mediated ISR simultaneously primed defense responses to nematode in soybean roots, we further examined the expression of PR1, PR3a, PR3b, PR5a, and NPR1-2 by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using gene-specific primer.

Soybeans were treated with Ba2-6, inoculated with SCN J2, and grown as previously described. Sterilized water was used as a control. To measure the transcript levels of defense-related genes in real time, soybean roots were collected at 0 and 3 days after nematode inoculation, froze immediately in liquid nitrogen, and stored at -80◦C until use. Total RNA was extracted from the soybean roots using the SV Total RNA Isolation Kit (Promega, Madison, WI, USA) and Total RNA from each sample (1 µg) was reverse-transcribed using the Prime Script RT Reagent kit (TaKaRa, China) according to the manufacturer’s instructions. The real-time qRT-PCR was performed on an Mx 3000P platform (Bio-Rad, Hercules, CA, USA). The 25-µL reaction mixtures contained 1.5 µL of 10 × diluted cDNA, 12.5 µL SYBR Premix Ex TaqTM II (Takara, Beijing, China), 9 µL double-distilled water, and 1 µL forward and reverse primers (10 µmol L−1) for selected genes. The gene-specific primer sequences are shown in Supplementary Table S1. The qRT-PCRs were denatured at 95 °C for 10 min and cycled 40 times at 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min. The qRT-PCR was performed on three biological replicates and each reaction was replicated three times. Actin11 was used as the internal reference gene for normalization, and the data were quantified using the 2−ΔΔCt method [48].

Statistical analysis

All data were analyzed using SPSS Statistics (Version 19.0; RRID: SCR_002865) and Microsoft Office Excel 2010. Significant differences were determined according to Duncan’s multiple range test (p < 0.05).

Results

Bacterial strains for antagonistic activity against H. glycines juvenile mortality

Based on the lethality test, we obtained and selected 8 bacterial strains from 76 isolates strains with better nematode inhibition. After 48 h of exposure, the antagonistic activity of Ba2-6, Ba2-4, Ba1-7, Ba4, Ba 17, Ba6, Ba11, and Ba2-8 as measured by a number of SCN J2 recovered ranged from 74.36 to 93.85% compared with that of the control. Among these, six isolates (Ba2-6, Ba2-4, Ba1-7, Ba7, Ba 4, Ba17, and Ba6) showed > 80% of juvenile mortality, Ba2-6 of which was the highest (93.85%), while 92.10% and 91.12% of juvenile mortality was recorded in Ba1-7 and Ba2-4. The lowest percentage of juvenile mortality was recorded in Ba2-8 (74.36%) (Table 1). Thus, the eight bacterial strains were selected for greenhouse and field analysis.

Table 1 Filtrate culture of bacterial isolates affected the J2s mortality of H. glycines
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Greenhouse experiment

At the 30th day after inoculation, soybean plants in the control had the largest number of females (236.43). Treatments with Ba2-4and Ba1-7 exhibited higher efficacies in reducing the number of females in soybean roots (69.92 and 67.97%, respectively), followed by Ba17 (40.82%) and Ba2-6 (38.39%) (Fig. 1). There was no significant difference between other treatments and the control. The results showed that four Bacillus isolates (Ba1-7, Ba2-4, Ba2-6, and Ba17) significantly reduced the number of females.

Fig. 1
figure 1

Effect of Bacillus on the control of adult females of H. glycines on soybean roots in pot experiments (n = 10). The values are mean ± standard error. CK denotes NB liquid medium. Means in each column followed by the same letter do not differ significantly according to Duncan’s multiple range test at P < 0.05

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The observation of shoot length, root length, and shoot fresh weight found no significant difference among all treatments (Table 2). Furthermore, seed treatment with Ba2-4, Ba2-6 and B4 significantly increased root fresh weight to 0.68, 0.68 and 0.56 g, corresponding to increases of 74.36, 74.36 and 43.59% above control treatment, respectively. There was no significant difference between other treatments and the control.

Table 2 Effect of bacterial isolates on plant growth parameters 30 days after inoculation with SCN
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Field experiment

Based on the strong control effect of Ba1-7, Ba2-4, Ba2-6, Ba2-8, Ba4, Ba6, Ba11, and Ba17 against H.glycines in the pot experiments, nematicidal activity was evaluated further in the field. Numerous large females were observed on the soybean roots of the control, while markedly fewer females were observed on the soybean roots after treating with Ba 1–7, Ba2-4, Ba2-6, and Ba4 in 2021, and with Ba 1–7, Ba2-4, Ba2-6, and Ba6 in 2022 (Fig. 2). In addition, the female number per plant was significantly reduced by 45.65% in 2021 and 44.96% in 2022 after Ba1-7 seed coating relative to non-treated control (Fig. 2). In contrast, seed treated with Ba2-8, Ba11, Ba6, and Ba17 had no significant effect on H.glycines with disease reductions in 2021 (Fig. 2A), Ba2-8, Ba4, Ba11, and Ba17 had no significant effect on H.glycines with disease reductions in 2022 (Fig. 2B). Therefore, Ba1-7, Ba2-4, and Ba2-6 exerted good and stable activity against H.glycines.

Fig. 2
figure 2

Effect of Bacillus on the control of adult females of H. glycines on soybean roots in field experiments in 2021 and 2022. The data in the figure are mean ± standard error, and different letters (a, b) above the bars indicate significant differences at P < 0.05 according to Duncan’s multiple range test (n = 30)

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Regarding plant growth parameters, after Ba2-6 treatment, the plant height and taproot length were significantly increased by 18.69% and 33.01% when compared to the control in 2021, and increased by 7.35% and 22.75% in 2022 (Table 3). Furthermore, taproot length was increased by 30.48% in 2021 and plant height was increased by 10.27% in 2022 after Ba2-4 treatment when compared to the control. However, analytical results of shoot fresh weight showed that there was no significant difference between the Bacillus strain treatments and the control, except that Ba11 and Ba4 in 2021 produced lower shoot fresh weights when compared with the control. Moreover, regarding the root fresh weight, Ba2-8 caused significantly higher root fresh weight than the control treatment in 2022, Ba1-7 caused significantly lower in 2021, and there was no significant difference between other treatments and the control.

Table 3 Effect of Bacillus on growth of soybean infected with H.glycines under field conditions in 2021 and 2022 (n = 30)
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The number of pods per plant significantly increased in plants treated with Ba2-4 (35.23%) and Ba2-8 (24.51%), and was significantly reduced in plants treated with Ba4(14.13%) compared to control plants in 2021(Fig. 3A). On the other hand, we found that the pod number per plant was not significantly different among Ba1-7, Ba2-4 and Ba2-6 and the blank control, while other treatments produced significantly higher pod numbers than the control in 2022 (Fig. 3B).

Fig. 3
figure 3

Effect of Bacillus treatments on pod number per plant of soybean infected with Heterodera glycines in field experiments in 2021 and 2022 (n = 30). The values are the mean ± standard error, and different letters (a, b) above the bars indicate significant differences at P < 0.05 according to Duncan’s multiple range test

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In the field experiments, the number of seeds per plant was significantly increased by 31.38%, 20.09%, 17.77% and 10% in the Ba1-7, Ba2-4, Ba2-6, and Ba2-8 treatments compared to the control, respectively in 2021 (Fig. 4A). The seeds per plant were significantly increased by 37.42%, 28.41%, and 25.16% after Ba1-7, Ba2-4 and Ba2-6 treatment when compared to control in 2022 (Fig. 4B). On the other hand, we found that the soybean 100-seed weight was not significantly different among these treatments in 2021 and 2022 (Fig. 5).

Fig. 4
figure 4

Effect of Bacillus treatments on seeds number per plant of soybean infected with Heterodera glycines in field experiments in 2021 and 2022 (n = 30). The values are the mean ± standard error, and different letters (a, b) above the bars indicate significant differences at P < 0.05 according to Duncan’s multiple range test

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Fig. 5
figure 5

Effect of Bacillus treatments on 100-seed weight of soybean infected with H. glycines in field experiments in 2021 and 2022 (n = 30). The values are the mean ± standard error, and the same letter does not differ significantly according to Duncan’s multiple range test at P < 0.05

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Identification of Bacillus species

Based on the lethality test, we obtained 8 bacterial strains from 76 isolates strains. The eight bacterial strains were selected for further greenhouse and field analysis. According to the results of the pot and field experiment, the three isolates- Ba1-7, Ba2-4, and Ba2-6 were selected from the eight strains for identification. The three bacterial isolates were subjected to morphological analysis (Supplementary Fig. S1) and biochemical characterization (Table 4). The three bacterial isolates were Gram-positive bacteria. Further, for physiological and biochemical characterization, all tested strains had positive reactions for citrate, D-xylose, D-Mannitol, L-Arabinose, PH 5.7, and Nitrate reduction and MR. All the strains were negative for the propionate. The gelatin liquefaction test indicated that only Ba2-4 could not liquefy gelatin. In addition, only Ba2-6 was negative, while the VP test had negative reactions in the amylohydrolysis test (Table 4).

Table 4 Physiological and biochemical characterization of bacterial isolates
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A phylogenetic tree was constructed using MEGA 7 software. BLAST analysis of 16 S rDNA sequence homology and phylogenetic analysis via the neighbor-joining method were conducted. The BLAST homologous search in GenBank indicated that Ba1-7 had 94% similarity to B. aryabhattai, and it was considered B. aryabhattai based on the phylogenetic tree constructed. Further, based on the morphological features and phylogenetic trees, strainBa2-4 was identified as B. megatherium and Ba2-6 as B. halotolerans (Fig. 6). These sequences were submitted to the GenBank database under accession numbers OL714327, OL714328, and OL714326, respectively.

Fig. 6
figure 6

Phylogenetic trees for isolates B. aryabhattai Ba1-7, B. megatherium Ba2-4, B. halotolerans Ba2-6, and known bacterial relatives, based on their 16 S rRNA gene sequences. The neighbor-joining method (NJ) was used to construct the trees. The numbers at the nodes represent percentages after 1000 bootstrap replicates

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Determination of protease production capacity of identified strains

The comparison of protease activity was shown in Fig. 7. The ratio of a transparent circle to colony diameter of strain Ba2-6 reached 2.01, indicating that Ba2-6 could produce protease.

Fig. 7
figure 7

Protease activity of strain Ba2-6

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Effect of Ba2-6 induces systemic resistance against H. glycines

While Ba2-6 resulted in low H. glycines mortality in the in vitro assay, it exhibited the capacity to control H. glycines in both the greenhouse and field conditions when used to coat soybean seeds, and the strain produces a large amount of protease. Therefore, we conducted a split experiment to verify whether Ba2-6 could elicit systemic resistance in soybeans against H. glycines. The split-root system showed that Ba2-6 induces systemic resistance to H. glycines in soybeans. Ba2-6 treatment caused a 74.29% reduction in total nematode penetration when compared with that in the control plants. Additionally, significantly fewer H. glycines J2, third-stage juveniles (J3), and fourth-stage juveniles (J4) were observed in the Ba2-6 treatment than in the control plants. Specifically, the numbers of J2, third-stage juveniles (J3), and fourth-stage juveniles (J4) were significantly reduced by 91.85%, 61.70%, and 75.32% in the responder roots of the Ba2-6 coated soybean, respectively, compared with that in the control (Fig. 8A). The proportions of J2, J3, and J4 in the Ba2-6 group were 12.72%, 84.34%, and 3.14%, respectively (Fig. 8B). Meanwhile, the proportions in the control group were 40.16%, 56.62%, and 3.27%, respectively. Therefore, the Ba2-6 treatment significantly decreased the proportions of J2 by 68.33%.

Fig. 8
figure 8

Effect of Ba2-6 on inducing systemic resistance to H. glycines using a split root system in soybean. (A) Effect of Ba2-6 on the penetration and development of H. glycines in the responder root system. (B) Effect of Ba2-6 on the proportion of each developmental stage of H. glycines in the responder root system. Numbers of second-stage juveniles (J2), third-stage (J3), and fourth(J4) in responder roots were counted 10 days after inoculation. Error bars represent standard deviations, and different letters (a, b) above the bars indicate significant differences at P < 0.05 according to a t-test (n = 4)

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To investigate whether Ba2-6-mediated ISR was accompanied by a defense response to salicylic acid or jasmonic acid pathways in soybeans, the expression levels of PR1, PR3a, PR3b, PR5a, and NPR1-2 in soybeans treated or not treated were measured. Regardless of the presence or absence of Ba2-6 treatment, there were no differences in the transcriptional levels of defense genes analyzed in unaffected soybeans. In contrast, 3 days after H. glycines inoculation, the expression of PR3b was nearly three-fold lower in Ba2-6 + SCN treatment than that in CK. Moreover, the expression levels of PR1, PR3a, PR5a, and NPR1-2 significantly increased 8.5-fold, 8.0-fold, 44.04-fold, and 3.43-fold in soybeans pretreated with Ba2-6 than in control soybeans, respectively (Fig. 9).

Fig. 9
figure 9

Induction of defense genes in soybean roots after H. glycines inoculation. Soybean root samples were taken at 0 and 72 h after H. glycines inoculation, and the expression levels of PR1, PR3a, PR3b, PR5a and NPR1-2 defense genes were analyzed by qRT-PCR. Error bars represent standard deviations; a t-test was used to determine significant differences between the Ba2-6-treated sample and the control (∗P < 0.05).

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Discussion

Soybean cyst nematodes, as important plant parasitic pests, are a devastating threat to soybean. It is urgent to develop alternative SCN control methods that are more environmentally friendly, safer for humans as well as meet the growing demand for food. In recent years, research on the application of bacteria to control SCN has drawn attention [18, 49,50,51]. In this study, we isolated Bacillus strains from the soil (46°41′ N, 125°35′ E), finding eight effective strains from 76 isolates against H. glycines for further evaluation. Further, through the combination of laboratory and field experiments, three strains of the eight isolates have the potential to be developed as a biocontrol agent to suppress H. glycines. Previous research also indicated that Bacillus spp. could be used to control PPN [52]. Further, numerous Bacillus strains have been treated as efficient alternatives to synthetic pesticides for SCN control in agriculture for the promotion of nematocidal potential and plant growth [13, 53]. Here, the results showed that the three bacteria belonged to Bacillus spp., B. aryabhattai (Ba1-7), B. megatherium (Ba2-4) and B. halotolerans (Ba2-6). Previous studies showed that the application of B. aryabhattai, B. megatherium, and B. halotolerans reduced the population of Meloidogyne incognita, H. glycines, and M. javanica on cotton [54], soybean plant [13], and tomato [49, 55], respectively. Therefore, our experimental results further confirmed that B. aryabhattai (Ba1-7), B. megatherium (Ba2-4), and B. halotolerans (Ba2-6) have a high potential to act as biocontrol agents of SCN, and this is the first comprehensive evaluation of the use of B. halotolerans as a biological control agent for H. glycines.

Previous studies have reported that Bacillus spp may be useful as a potential source of biocontrol agents. For example, B. halotolerans strain LYSX1 showed 100% juvenile mortality after 12 h exposure, decreasing the number of egg masses and galls but increasing the tomato root and shoot fresh weight in vitro [55]. Further, B. megaterium showed significantly increased mortality of SCN J2 compared to the blank control at 12 h and 48 h [21]. Our evaluation of the three strains Ba1-7, Ba2-4, and Ba2-6 had a negative effect on H. glycines, the results of the present study agree with previous studies [49]. It was demonstrated that they have nematocidal potential against H. glycines. Additionally, as described in previous experiments, B. aryabhattai, B. megatherium, and B. halotolerans could inhibit egg hatching, affect nematode juveniles, reduce the number of cysts, or suppress the galling index [13, 22, 49]. Thus, it can be concluded that the B. aryabhattai, B. megatherium, and B. halotolerans have high potential for controlling SCN.

Most bacteria reduce nematode populations by metabolic products, such as toxins and enzymes, or induce systemic resistance. Nematocidal activity was related to the secondary metabolites, such as antibiotics, enzymes, and toxic compounds, as shown by previous studies [56,57,58]. Many bacteria belonging to the genus Bacillus can excrete large amounts of enzymes into culture mediums. Some proteases can degrade and destroy the nematode body wall, such as serine protease and collagenase. It has been reported that Brevibacillus laterosporus strain G4 degrades nematode cuticles, and the involvement of hydrolytic proteases as potential nematoxic components in the bacterial infection of nematodes [59]. An alkaline protease BLG4 with strong nematocidal activity was pureed from B. laterosporus G4, and the BLG4-de efficient mutant showed a sharp drop in nematocidal and cuticle-degrading activities [60]. Our experimental results also showed that the Ba2-6 strain produced protease. However, the nematocidal secondary metabolites produced by B. halotolerans (Ba2-6) have not been characterized.

The results of the split-root system showed that Ba2-6 induces systemic resistance to H. glycines and resulted in significantly reduced penetration in soybeans. Previous studies have shown that S. fredii Sneb183 [19], B. subtilis [46], and Microbacterium maritypicum Sneb159 [51] reduced H. glycines penetration by ISR. Moreover, according to the results of the present study, Ba2-6 treatment caused a 74.29% reduction in the total nematode penetration and significantly fewer J2 H. glycines when compared with that in the control plants. However, why the proportion of J3 H. glycines in Ba2-6-treated soybean was significantly higher than that in the control is unknown. This may be because only one sampling and staining survey was conducted, and the developmental dynamics of nematodes were unknown. So, in the next step, we will further explore the impact of Ba2-6 treatment on the developmental dynamics of H. glycines. Perhaps one of the mechanisms is that Ba2-6 reduced the total number of juveniles and lengthened SCN development time.

PR1, PR3a, PR3b, PR5a, and NPR1-2 genes were associated with plant-induced disease resistance. In our study, 72 h after inoculation with H. glycines, the transcript levels of PR1, PR3a, PR5a, and NPR1-2 increased, and that of PR3b was not altered in Ba2-6-pretreated soybeans compared with those in nontreated soybeans. Another study reported that 48 h after inoculation with H. glycines, the expression of the PR1 and PR5 genes were increased significantly, and PR3b was decreased, and systemically in Klebsiella pneumoniae pretreated soybeans [12, 61]. It was reported that the expression of PR5, PR2, and PR1 was induced, and that of PR3 was not altered in roots During H. schachtii infection of Arabidopsis [62]. Further, the expression of NPR1-1 in Sneb207 pretreated soybeans was increased compared to CK [13]. Thus, Ba2-6-induced soybean resistance to H. schachtii may be associated with these genes. Furthermore, Dependence on both SA and JA/ET signaling pathways to induce plant defense against H. glycines has also been reported [12]. Therefore, our results suggested that Ba2-6 might also activate SA and JA/ET signaling pathways to induce plant defense against H. glycines.

In summary, our experimental results suggested that soybean seeds with the treatments of Ba1-7, Ba2-4, and Ba2-6 significantly (p < 0.05) reduced the number of females of H. glycines in soybean under both greenhouse and field conditions. We also showed that the three strains decreased the activity of J2 in vitro. Moreover, Ba2-6 produced protease and induced local and systemic resistance, which suppressed nematode infection and induced the expression of defense-related genes involved in the SA and JA signaling pathways. Thus, these findings suggest that B. aryabhattai (Ba1-7), B. megatherium (Ba2-4), and B. halotolerans (Ba2-6) have the potential to control H. glycines. This study thus provides strong support for the further exploration of the use of Bacillus spp. as biocontrol agents to control soybean cyst nematodes in crops. This study is the first report of B. halotolerans Ba2-6 eliciting ISR against SCN. Thus, our results suggest B. halotolerans as a potential biocontrol agent for SCN.

Availability of data and materials

Data is provided within the manuscript or supplementary information files.

Abbreviations

SCN:

Soybean cyst nematode

PPN:

Plant-arasitic nematodes

PR:

Pathogenesis-related

SA:

Salicylic acid

JA:

Jasmonic acid

ET:

Ethylene

ISR:

Induced systemic resistance

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Contributions

Y.Z. wrote the main manuscript text and revised it. J.C. reviewed the main manuscript text. P.X. and Y.F. investigated the data, J.L.investigated and analyzed the data. L.C. and Y.G. designed of the work. All authors reviewed the manuscript.

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This work was supported by the Scientific Research Initiation Program of Returning and Introducing Talents in Heilongjiang Bayi Agricultural University (XYB201904).

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  1. Yuanyuan Zhou and Jingsheng Chen contributed equally to this work.

Authors and Affiliations

  1. College of Agronomy, Heilongjiang Bayi Agricultural University, Daqing Heilongjiang, China

    Yuanyuan Zhou, Jing Li & Yongxia Guo

  2. Heilongjiang Provincial Key Laboratory of Crop-Pest Interaction Biology and Ecological Control, Heilongjiang Bayi Agricultural University, Daqing, China

    Yuanyuan Zhou

  3. College of Biology and Food Engineering, Chongqing Three Gorges University, Chongqing, Sichuan, China

    Jingsheng Chen

  4. Tobacco Research Institute of Chinese Academy of Agricultural Sciences, Qingdao, Shandong, China

    Yaxing Feng

  5. Heihe Branch, Heilongjiang Academy of Agricultural Sciences, Heihe, Heilongjiang, China

    Peng Xiang

  6. College of Plant Protection, Nematology Institute of Northern China, Shenyang Agricultural University, Shenyang, Liaoning, China

    Lijie Chen

  7. Key Laboratory of Low-carbon Green Agriculture in Northeastern China, Ministry of Agriculture and Rural Affairs P. R., Daqing, China

    Yongxia Guo

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Y.Z. Writing - review & editing, Validation, Writing-original draft. J.C. Writing-review, P.X. and Y.F. Investigation, J.L. Investigation and Formal analysis. L.C. Conceptualization, Y.G. Editing, Supervision, Funding acquisition.

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Zhou, Y., Chen, J., Feng, Y. et al. Biocontrol Potential of Bacillus strains against soybean cyst nematode (Heterodera glycines) and for promotion of soybean growth. BMC Microbiol 24, 371 (2024). https://doi.org/10.1186/s12866-024-03514-y

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